Ubiquitin specific proteases (USP) are cysteines proteases which belong to the deubiquitinating enzymes (DUBs) family.
Deregulation of the ubiquitin-proteasome system has been implicated in the pathogenesis of many human diseases, including cancer (Hoeller et al. Nat Rev Cancer 2006, 6(10), 776-788), neurodegenerative disorders (Rubinsztein, Nature 2006, 443(7113), 780-786) and viral diseases (Gao & Luo Can J Physiol Pharmacol 2006, 84(1), 5-14). The market success of the proteasome inhibitor Velcade® (bortezomib) for the treatment of multiple myeloma and mantle cell lymphoma has established this system as a valid target for cancer treatment (Adams, Nat Rev Cancer 2004, 4(5), 349-360). A promising alternative to targeting the proteasome itself would be to interfere with the upstream ubiquitin conjugation/deconjugation machinery, to generate more specific, less toxic anticancer agents.
Mono- and polyubiquitination can be reversed by deubiquitinating enzymes, which specifically cleave the isopeptide bond at the C-terminus of ubiquitin. Ubiquitin specific proteases and ubiquitin C-terminal hydrolases (UCH) enzymes are the best characterized members of the DUB family (Komander et al. Nat. Rev. Mol. Cell Biol. 2009, 10(8), 550-63; Nijman et al. Cell 2005, 123(5), 773-786). UCHs are thought to cleave small protein substrates preferentially and to be involved principally in the processing and recycling of ubiquitin, but their specific functions remain poorly understood. USPs constitute the largest subfamily of DUBs, with more than 60 members. They remove ubiquitin from specific protein substrates, thus preventing their targeting to the proteasome or regulating their subcellular localization and activation (Daviet & Colland, Biochimie 2008, 90(2), 270-83). USPs are emerging as potential targets for pharmacological interference with the ubiquitin regulation machinery, based on their protease activity and involvement in several human diseases (Colland, Biochem Soc Trans 2010, 38, 137-43).
USP7 (Ubiquitin Specific Protease 7)/HAUSP (Herpes Associated Ubiquitin Specific Protease) is a 135 kDa protein of the USP family. USP7 has been shown to interact with viral proteins, such as ICP0 (Vmw 110), a herpes simplex virus immediate-early gene stimulating initiation of the viral lytic cycle (Everett et al., J Virol 73, 1999, 417-426), and EBNA1 (Epstein-Barr Nuclear Antigen-1) (Holowaty et al., J Biol Chem 2003, 278, 29987-29994 and 47753-47761). Human proteins, such as p53 and the major E3 ligase of p53, Mdm2, have also been identified as partners and substrates of USP7 (Cummins et al. Nature 2004, 486, Cummins & Vogelstein, Cell Cycle, 2004, 3, 689-692; Li et al. Mol Cell 2004, 13, 879-886; Li et al. Nature 2002, 416, 648-653). More generally USP7 can deubiquitinate different targets, including Mdm2 and p53, and the net deubiquitination of these latter targets ultimately determines functional p53 levels. Consistent with recent reports, USP7 silencing has also been shown to increase steady-state p53 levels by promoting Mdm2 degradation. Binding of USP7 to p53 was recently shown to be regulated by TSPYL5, a protein potentially involved in breast oncogenesis through a competition with p53 for binding to the same region of USP7 (Epping et al., Nat Cell Biol. 2011, 13(1):102-8). More recently, both upregulation and downregulation of USP7 have been shown to inhibit colon cancer cell proliferation in vitro and tumor growth in vivo, by resulting in constitutively high p53 levels (Becker et al. Cell Cycle 2008, 7(9), 1205-13).
USP7 also alters the level of the p16INK4a tumor suppressor through Bmi1/Me118 stabilization (Maertens et al., Embo J. 2010, 29, 2553-2565). Additional proteins involved in genomic integrityregulation such as the DNMT1 DNA methylase and the Claspin adaptor are also stabilized by USP7 (Du et al., Science Signaling 2010, 3(146):ra80; Faustrup et al., J. Cell Biol. 2009, 184(1):13-9). Importantly, the abundance of USP7 and DNMT1, a protein involved in maintaining epigenetic methylation required to silence genes involved in development and cancer, correlates in human colon cancer (Du et al., Science Signaling, 2010, 3(146):ra80). USP7 has also been shown in human cells to deubiquitinate the well-known tumor suppressor gene PTEN, which provokes its nuclear export and hence its inactivation (Song et al., Nature 2008, 455(7214), 813-7). More importantly, USP7 overexpression was reported for the first time in prostate cancer and this overexpression was directly associated with tumour aggressiveness (Song et al., Nature 2008, 455(7214), 813-7).
USP7 has also been shown in human cells to deubiquitinate FOXO4, which provokes its nuclear export and hence its inactivation; consequently the oncogenic PI3K/PKB signaling pathway was activated (van der Horst et al., Nat Cell Biol. 2006, 8, 1064-1073) Finally, USP7 plays an important role in p53-mediated cellular responses to various types of stress, such as DNA damage and oxidative stress (Marchenko et al., Embo J. 2007 26, 923-934, Meulmeester et al., Mol Cell 2005, 18, 565-576, van der Horst et al., Nat Cell Biol. 2006, 8, 1064-1073).
Synthetic inhibitors of USP7 protein binding containing the polypeptide portion P1-Gly-P3-Ser, where P1 is a glutamic acid residue or an amino acid with a non polar side chain and P3 is a glycine residue or an amino acid with non polar side chain, have been reported (WO2006072048).
The phenotypes associated with USP7 silencing and the known connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and PI3K/PKB pathways, strongly suggest that targeting USP7 with small-molecule inhibitors may be beneficial in the treatment of cancers and viral diseases (Sippl et al., Future Oncology 2011, 7, 619-32). Inhibitors against USP7 were recently reported (Colland et al. Molecular Cancer Therapeutics 2009, 8, 2286-95 and EP 1 749 822 and PCT/EP2011/050523.2).
However, to date, no specific and reversible USP7 small molecule inhibitors seem to have been reported.